It is widely hypothesized that memory is stored in the brain as enduring changes in the wiring diagram and strength of synaptic connections between neurons. The receptive structure for most of these synapses is the dendritic spine, a micron-scale protrusion emitted from the neuron's dendrite which senses neurotransmitter released by the closely-opposed presynaptic terminal. Several lines of evidence suggest that dendritic spine density and turnover in the neocortex is modified by learning and is positively correlated with learning rate. Since the early 1990s, it has been established that dendritic spine density varies by about 35% over the course of the ovarian cycle in female rats, mice and nonhuman primates, being higher in proestrus and diestrus and lower in estrus. Similarly, surgical ovariectomy leads to an ~40% loss of spine density which can be rapidly reversed by 17-beta-estradiol (E2) treatment. These findings have suggested an important question: If memory is largely encoded in spiny synapses and if spine density fluctuates by ~40% over the ovarian cycle, then how does long-term memory persist in female mammals in the face of this fluctuation? Why doesn't memory degrade with each ovarian cycle? To date, measurements of spines in relationship to the ovarian cycle have relied upon traditional anatomical methods applied to postmortem tissue. These methods preclude within-animal and within-dendrite comparisons across time. Here, we propose to use in vivo two-photon microscopy together with existing transgenic mouse lines (including Thy1M-EGFP for sparse labeling of layer 5 pyramidal cells) to produce time-lapse images of identified spiny dendrites in the neocortex and thereby address two crucial questions. Aim 1: When spines are lost following ovariectomy, do new spine regrow in those same dendritic locations when estrogen levels rise following E2 treatment? Pilot studies will use traditional histological techniques to determine the regions of the neocortex in which the largest and most reliable loss/recovery of spines can be seen in layers I - III. This information will then be used to guide the placement of cranial windows for time-lapse imaging spanning overiectomy or sham surgery and subsequent E2 or vehicle treatment. Aim 2: When spines are lost during estrus do they tend to regrow in that same dendritic location when estrogen levels rise again in proestrus/diestrus? As in Aim 1, initial histological experiments will guide the placement of cranial windows. Then, daily monitoring will commence together with E2 ELISA and vaginal swab assays of ovarian cycle status. Time-lapse imaging of dendritic spines together with manipulation of estrogen levels either though exogenous manipulation (Aim 1) or natural cycles (Aim 2) will allow us to determine whether dendritic spines lost during low-estrogen conditions are regrown at the same dendritic location when high estrogen levels return. Imaging over several cycles (Aim 2) will allow us to determine if spine loss in estrus tends to occur in a particular subset of spine locations or spine types that define a high turnover pool. Finally, we will use Thy1M-GFP mice injected with virus encoding red fluorophore in posteromedial thalamus as a first attempt to determine whether regrown spines are re-contacting their original thalamo-cortical axons in neocortical layer 1. This work shall have important clinical implications for hormone replacement therapy after ovariectomy or menopause.